The goal of the research described in this proposal is to contribute to a detailed understanding of the functional mechanism of a prokaryotic two- component signaling system. We are particularly interested in the mechanism of histidine kinase function as it is exemplified by CheA, the sole histidine kinase controlling bacterial chemotaxis in E. coli and S. typhimurium. We will explore the fundamental properties governing CheA kinase regulation by both intramolecular and intermolecular mechanisms. Intramolecular regulation mechanisms will be elucidated through a combination of molecular biology and biochemical/biophysical techniques and will be aimed at understanding how relative domain motions and intramolecular binding events affect CheA's autophosphorylation activity. Intermolecular kinase regulation mechanisms will be investigated, again, through a combination of molecular biology and biophysics. We aim to add to the knowledge about how other proteins in the signaling pathway controlling bacterial chemotaxis interact with CheA to affect its kinase activity. Particularly, we are interested in the roles of the transmembrane chemoreceptors and the CheW coupling protein, which are in a stable complex with CheA during signaling and adaptation and which may alter its activity via various conformational changes. To this end, it is a goal of this project to crystallize the ternary complex of the chemoreceptor cytoplasmic domain, CheW, and CheA using the more-stable Thermotoga maritima homologs of these proteins.